This project is intended to provide a detailed understanding of a recently discovered prokaryotic adaptive immune system known as CRISPR (clusters of regularly interspaced short palindromic repeats). CRISPR drives adaptation to harmful invading nucleic acids - such as conjugative plasmids, transposable elements and phages - using an RNA-mediated interference (CRISPR interference) mechanism with fundamental similarities to our innate and adaptive immune responses. CRISPR-Cas defense systems have been identified in 88% of archaeal genomes and 39% of bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis. It has been shown to modulate the horizontal gene transfer and biofilm formation. Although the details of this defense mechanism remain to be determined, two distinct stages are recognized: (i) adaptation upon first exposure to the foreign nucleic acid whereby some combination of CRISPR-associated (Cas) proteins extracts recognizable features from the genomes of viruses (bacteriophages) and plasmids as protospacers that are subsequently incorporated as spacers at the 5' end of genomic CRISPR loci; and (ii) interference upon re-exposure to the same nucleic acid whereby a ribonucleoprotein complex comprised of small guide RNAs (crRNA) derived from genomic CRISPRs and different Cas proteins targets foreign nucleic acids for destruction. The lack of information on the molecular and structural properties of the Cas proteins and complexes severely impedes progress in the study of CRISPR mediated bacterial immunity. The proposed research is based on the successful structure determination of several important Cas proteins and the successful reconstitution of the Type I-C Cascade complex from B. halodurans. In this proposal, we propose experiments to understand the CRISPR interference mechanism in Type I-C CRISPR-Cas system. We build upon strong preliminary data to (1) characterize the structure-function of individual components of the Type I-C Cascade, (2) establish function assays and determine the EM and crystal structure of the intact I-C Cascade, and (3) characterize the structure-function of the Cascade-interacting protein Cas3, an essential factor in all Type I CRISPR-Cas systems. Our findings will serve to reveal the common theme and mechanistic diversity among different CRISPR-Cas systems.